The goal of this ongoing research program is to understand how eukaryotic cells are able to transduce intracellular calcium signals into biological responses. The general scientific questions being addressed are: how are calcium signal transduction complexes encoded, made, assembled and able to convert changes in the concentration of ionized calcium into a biological response? The approach is interdisciplinary, with an emphasis on perturbation/mutant analysis. Mutant analysis in this sense includes biochemical analysis of mutant organisms that have defects in calcium mediated cellular responses, site-specific mutagenesis and protein engineering studies of physiologically important calcium signal transduction complexes, and analysis of organisms that have been perturbed by the introduction of mutant genes or proteins that encode these signal transduction activities. The focus continues to be on calmodulin (CaM), a ubiquitous calcium binding protein that exists as an integral subunit of several macromolecular complexes, including enzymes, cytoskeletal structures and membrane-resident transport systems. CaM is a member of a class of regulatory proteins, sometimes referred to as effector proteins, that modulate the functional properties of other proteins. Because CaM must be complemented by a parallel analysis of calmodulin binding proteins. This proposal focuses on the basic mechanisms of CaM-regulated protein kinase activity, because of the generality of protein phosphotransferase activity in signal transduction and the clear in vivo role of one kinase, the non- muscle/smooth muscle myosin light chain kinase, as an initiator of cellular responses to calcium signals. The proposed studies have the potential of providing insight into how CaM- mediated signal transduction complexes are encoded, assemble into supramolecular complexes, and transduce calcium signals into cellular responses. By combining detailed in vitro analyses of physiologically relevant protein complexes with in vivo studies that seek to screen for perturbations of cell physiology, there is the potential of gaining insight into fundamental questions about eukaryotic cell signal transduction. In the longer term, there is the clear potential of developing a more rational basis for new drug design, understanding the molecular basis of disease states, and providing a mechanistic basis for therapy.